Endocannabinoids are lipid mediators that exhibit analgesic, neuroprotective, and anti-inflammatory activities through cannabinoid receptors. The two most studied endocannabinoids are derivatives of arachidonic acid (AA) - i.e., 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (AEA). AA is a substrate for the cyclooxygenase enzymes (COX-1 and COX-2), which carry out the committed step in prostaglandin (PG) biosynthesis. We discovered that 2-AG and AEA are oxygenated by COX-2 much more efficiently than by COX-1 and, like AA, are oxygenated to intermediates that are converted into glycerol ester and ethanolamide analogs of PGs (i.e., PG-Gs and PG-EAs). These analogs exert potent biological activities that are independent of classical PG receptors. Thus, endocannabinoids may be the substrates for a COX-2-selective signal transduction pathway. Oxygenation by the COX enzymes is inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs), which contributes to their pharmacological activity. Very recently, we discovered that NSAIDs which are classified as weak, reversible inhibitors of AA oxygenation are potent, poorly reversible inhibitors of 2-AG and AEA oxygenation. This "substrate-selective" inhibition provides a mechanism by which endocannabinoid metabolism through COX-2 can be inhibited without any impact on AA metabolism. Particularly exciting is our discovery that (R)-enantiomers of arylpropionic acid NSAIDs (e.g., (R)-flurbiproten), which were previously thought to be inactive against COX enzymes, are efficient inhibitors of 2-AG oxygenation by COX-2 in vitro and in cultured dorsal root ganglion cells. This finding may explain the analgesic activity of (f?)-flurbiproten in humans and in animal models of neuropathic pain. The latter has been associated with elevation of endocannabinoid levels in the spinal cord. We propose to elucidate the molecular determinants of substrate-selective inhibition of COX-2 by (R)-NSAIDs using a combination of site-directed mutagenesis, structure-activity analysis, and X-ray crystallography. We will use this information to optimize the potency and selectivity of this class of agents and evaluate the most promising compounds in dorsal root ganglion cells and the chronic constriction model of neuropathic pain. We will also identify the enzyme(s) that hydrolyze PG-Gs to PGs, thereby limiting their half-lives and biological activities. This will provide new insights into NSAID action and tools and drug candidates focused on the COX-2-endocannabinoid metabolism and signaling pathway.